Swine dysentery is a highly contagious disease of growing and finishing swine which has a significant economic impact on the United States swine industry. Although the causative agent has been known for nearly 20 years, no effective measures, other than medication of animals and sanitation of premises, are available to prevent the occurrence of the disease or reduce its severity once introduced into a susceptible herd. Recently published data from Iowa State University indicated a projected statewide cost for prevention and control of swine dysentery of approximately $2.4 million per month. Since the Iowa swine population represents one-fourth of the nation's industry, the annual losses due to swine dysentery for the United States may represent as much as $115.2 million. Owen, Iowa State J. Res., 62:293-311 (1987).
Serpulina hyodysenteriae is the primary etiologic agent of swine dysentery. Harris and Lysons, Diseases of Swine, 7th ed., Iowa State University Press (Ames, Iowa), at pp. 599-616 (1992); and Stanton et al., Int. J. Syst. Bacteriol., 41:50-58 (1991). Nine serotypes of S. hyodysenteriae have been recognized worldwide, with serotypes 1 and 2 being the most prevalent in the United States. Baum and Joens, Infect. Immun., 25:792-796 (1979); Mapother and Joens, J. Clin. Micro., 22:161-164 (1985); and Li et al., J. Clin. Microbiol., 29:2794-2797 (1991). The diagnosis of swine dysentery is based on herd history, clinical signs, observation of characteristic intestinal lesions, and isolation of S. hyodysenteriae from feces or intestine using selective agar medium incubated anaerobically for 2 to 4 days. Chengappa et al., Resort of the Committee on Swine Dysentery, American Association of Veterinary Laboratory Diagnosticians, Inc. (Columbia, Mo.) (1989). Laboratory confirmation of S. hyodysenteriae by culture is based upon colony morphology, pattern and intensity of hemolysis, and other growth characteristics, all of which are very similar for the non-pathogenic Serpulina innocens, a common inhabitant of the colon of swine. Kinyon and Joens, (1979). As a result, a definitive diagnosis of swine dysentery can be very challenging particularly when the disease occurs on premises where weakly .beta.-hemolytic intestinal spirochetes (WBHIS) are present in the swine population.
An important aspect of swine dysentery is the occurrence of prolonged shedding of S. hyodysenteriae in the feces of some animals following recovery from diarrhea. Songer and Harris, Am. J. Vet. Res., 39:913-916 (1978); and Fisher and Olander, Am. J. Vet. Res., 46:450-455 (1981). Asymptomatic carrier-shedder swine are important reservoirs for maintenance of S. hyodysenteriae on infected premises and transmission of the organism to uninfected premises. The solution to swine dysentery prevention lies in being able to quickly and accurately identify carrier-shedder swine and avoid their entry into uninfected herds. However, identification of asymptomatic carrier-shedders of S. hyodysenteriae, is difficult due to the detection limits of currently available laboratory isolation procedures.
Direct culture of diagnostic specimens is the only method available for laboratory identification of S. hyodysenteriae. However, it is well known that the sensitivity of the direct culture method depends upon the number of organisms present in the sample, which in turn depends on the stage of infection of the animal at the time of collection. Kunkle and Kinyon reported that the numbers of S. hyodysenteriae in porcine colonic contents at the onset of swine dysentery ranged between 2.times.10.sup.6 and 2.times.10.sup.10 CFU/g when cultured using the selective BJ medium. Kunkle et al., J. Clin. Microbiol., 26:2357-2360 (1988). In contrast, subclinically affected animals may shed recoverable numbers of spirochetes only sporadically and in much lower numbers than animals with clinical swine dysentery often resulting in false negative culture results. Field cases of swine dysentery also may contain drug residues that adversely affect recovery of viable S. hyodysenteriae by culture.
Identification of S. hyodysenteriae by culture is highly subjective and can lead to false results, particularly when results of cultures are interpreted by inexperienced laboratory workers. For this reason, several biochemical tests have been proposed for rapid differentiation of enteropathogenic and non-pathogenic intestinal spirochetes of swine. Achacha et al., J. Vet. Diag. Invest., 3:211-214 (1991); Belanger et al., J. Clin. Microbiol., 29:1727-1729 (1991); Hunter et al., Vet. Rec., 104:383-384 (1979); and Smith et al., Vet. Microbiol., 24:29-41 (1990). Although these biochemical characteristics are highly conserved among field isolates of S. hyodysenteriae, WBHIS have been shown to yield highly variable results making a conclusive identification of S. hyodysenteriae based on biochemical tests alone practically impossible. Achacha et al, cited supra; Belanger et al., cited supra; Burrows et al., Vet. Rec., 108:187-189 (1981); Kinyon et al., Infect. Immun., 15:638-646 (1977); Lymbery et al., Vet. Microbiol., 22:89-99 (1990); Picard et al., Can. J. Microbiol., 26:985-991 (1980); Ramanathan et al., Vet. Microbiol., 37:53-64 (1993); and Torp and Thorensen, Proc. 12th Congr. Int. Pig Vet. Soc., The Hague, The Netherlands, at page 270. (1992). In addition, the biochemical tests require growth of the organism for 2 to 4 days.
Other methods of differentiating S. hyodysenteriae from WBHIS include growth inhibition by discs soaked in antiserum (Lemcke and Burrows, Vet. Rec., 104:548-551 (1979)) and rapid slide agglutination (Burrows and Lemcke, Vet. Rec., 108:187-189 (1981)). In addition to problems of non-specific clumping of spirochetes in the saline control in the slide agglutination test, these tests require large numbers of pure culture of spirochetes which can take up to 3 weeks to grow. Lysons, Vet. Rec., 129:314-315 (1991). Although pre-absorption of reference polyclonal antisera with WBHIS increases the specificity of the serological tests, occasional S. hyodysenteriae isolates continue to be falsely classified as non-pathogenic in these tests. An alternative method using microscopic agglutination under phase contrast or dark field microscopy was recently proposed. However, some isolates of S. hyodysenteriae gave weaker reactions in that assay than with the slide agglutination test (Lysons, cited supra).
Mouse monoclonal antibodies capable of differentiating S. hyodysenteriae from porcine WBHIS have also been proposed as potential diagnostic reagents. Sellwood et al., Proc. 12th Congr. Int. Pig Vet. Soc., The Hague, The Netherlands, at page 264 (1992); and Thomas and Sellwood, J. Med. Microbiol., 37:214-220 (1992). However, other studies, indicate that spirochetes other than S. hyodysenteriae can express antigenic determinants recognized by these reagents and cause false positive results. Taylor et al., Proc. 12th Congr. Int. Pig Vet. Soc., The Hague, The Netherlands, at page 280 (1992). The fact that no serological reagents are available commercially also limits the applicability of serological techniques to routine diagnosis of swine dysentery.
Certain genes encoding S. hyodysenteriae antigens and capable of eliciting protection against infection in mice have been cloned and expressed in Escherichia coli using a phage expression system. Boyden et al., Infect. Immun., 57:3808-3815 (1989). However, none of these reagents have been examined for potential application as diagnostic tools. One of the most recent diagnostic applications of recombinant DNA technology to swine dysentery control used oligodeoxynucleotide probes to 16S rRNA of S. hyodysenteriae. Jensen et al., J. Clin. Microbiol., 28:2717-2721 (1990). However, the sensitivity of this probe method for detection of spirochetes in feces was equivalent to routine bacteriological culture (10.sup.5 organisms/g of feces), and further studies question the specificity of the 16S rRNA probe to S. hyodysenteriae (Torp and Thoresen, cited suora). Dot blot hybridization with whole-chromosomal probes and DNA probes for identification of S. hyodysenteriae have been reported. Combs and Hampson, Res. Vet. Sci., 50:286-289 (1991); Sotiropoulos et al., J. Clin. Microbiol., 31:1746-1752 (1993); and Sotiropoulos et al., J. Clin. Microbiol., 32:1397-1401 (1994). Although the sensitivity of the whole-chromosomal probes was not reported, colony dot blot hybridization with DNA probes was shown to be only slightly better than culture (10.sup.4 organisms/g of feces). These tests are labor intensive, require specialized equipment, and have turn-around times that are incompatible with routine laboratory diagnosis.
A solution to prevention of disease caused by S. hyodysenteriae lies in being able to quickly and accurately identify carrier-shedder animals and avoid their entry into uninfected herds. Therefore, there is a need to develop a method and reagents for detecting S. hyodysenteriae in low numbers specifically, rapidly and directly from diagnostic and environmental samples. There is also a need to develop a sensitive and specific method for rapid detection of S. hyodysenteriae in a biological sample to diagnose and monitor infection in acutely- or subclinically-infected animals before, during and after treatment and in their environment. There is also a need to develop a method for rapid detection of S. hyodysenteriae for monitoring disinfection of the environment in contact with infected animals.